Our long range goal is to understand the role of phospholipids in the assembly, organization, and function of the mitochondrial membrane. Toward this end, we have developed the tools to genetically manipulate the levels of the mitochondria-specific phospholipid cardiolipin (CL) through cloning of the gene encoding L synthase and generation of a null mutant. Previous in vitro studies had suggested that CL was critical to the function of several mitochondrial enzymes. However, until now, it has been impossible to extend these signals to reveal a role for CL in vivo. We now have the molecular tools to do so. Our lab was the first to publish the cloning of the CRD1 gene (previously called CLS1) encoding CL synthase, and the purification of the two key enzymes of the CL pathway, phosphatidylglycerolphosphate synthase (the PGS1 gene product) and CL synthase. We constructed a crd1 null mutant which has no detectable CL in its membranes. The crd1 mutant can grow on both fermentable and non-fermentable carbon sources at 30 degrees Centigrade, but cannot grow at 37 degrees Centigrade. With its lack of CL and conditional lethality, the mutant is a powerful tool with which to carry out in vivo studies of CL function. In this proposal, we seek to understand the function of CL, and how CL synthesis is regulated. Proposed experiments will address the following questions: 1. What is the role of CL in mitochondrial function and cell viability? We will use the genetic approach of isolating suppressors of the temperature sensitivity phenotype of the crd1 null mutant, and characterizing the suppressor genes to understand why CRD1 is essential at elevated temperatures. In addition, we will compare the crd1 null mutant and isogenic wild type with respect to oxidative phosphorylation, mitochondrial membrane potential, and function of the mitochondrial permeability transition pore. 2. How is expression of the CL structural genes regulated? We will use northern blot analysis and fusion to reporter genes to determine how PGS1 expression is regulated. In addition, we will determine if Pgs1p or Crd1p are controlled post-translationally. Finally, we will identify regulatory genes which affect CL synthesis and determine how they interact to control expression of the structural genes 3. How is CL synthase activity regulated in the mitochondrial membrane? We have shown that CL synthase is part of a large complex in the mitochondrial membrane We will use both the yeast two-hybrid screen and chemical cross-linking to identify components of the complex which interact with L synthase. We will then determine how deletion or over- expression of these components affects CL synthase activity. CL is the major polyglycerolphospholipid in the mammalian heart, comprising 15% of the total cardiac phospholipid mass. Because CL is crucial for many aspects of mitochondrial function, the results of the proposed experiments will provide information critical to understanding how the heart functions.